In the fields of medicine, biomedical research and diagnostic and clinical chemistry, there is often a need to visibly detect a cell or component thereof in order to effectively examine, diagnose or treat an animal or person. Many colorimetric and fluorometric compounds have been used for such detection. For example, copending and commonly assigned U.S. Ser. No. 824,757, filed Jan. 31, 1986, now U.S. Pat. No. 4,803,161, by Babb et al describes novel phenalenone and benzphenalenone fluorescent compounds and reducible precursors which are useful in various medical and diagnostic applications, such as for staining cells and for detecting microorganisms.
Many studies and determinations of physiologically reactive species (that is, species which will react in a specific binding reaction with a complementary binding partner, such as cell components, proteins, peptides, polypeptides, carbohydrates, nucleic acids, haptens and other materials known in the art) are carried out using "labels" which facilitate the detection or separation of the materials of interest at low concentrations. In one such application, the diagnosis of pathological conditions and the detection of drugs or narcotics is often carried out using labeled materials in specific binding assays using competitive binding principles.
Whenever labels are used, sensitivity is of prime importance due to the generally low levels of detectable species present. Radiometric labels have several drawbacks including limited sensitivity, short useful life and handling hazards. Various colorimetric labels can be incorporated into latex particles for use in analytical methods, but the dyes may be hard to detect and provide limited sensitivity.
Fluorescence spectroscopy, one of the most sensitive and versatile of the optical analytical techniques, has become increasingly popular in recent years to overcome the drawbacks of other labeling techniques. Fluorescent labels for immunoassays are described in U.S. Pat. Nos. 4,259,313 (issued Mar. 31, 1981 to Frank et al) and related 4,283,382 (issued Aug. 11, 1981 to Frank et al) which describe labeled polymeric particles useful in immunoassays.
After appropriate excitation, many known fluorescent compounds used in biological methods emit radiation at a wavelength less than 600 nm. In such cases, detection of the component of interest can be interfered with by other components commonly found in biological samples and which emit electromagnetic radiation at or near the emission wavelength of the fluorescent compound. To overcome this, various filtration steps can be carried out to remove interferents but this results in a tedious and costly procedure. It would be highly desirable to have fluorescent compounds which are detectable at a wavelength at or above about 600 nm. It would also be useful to have such compounds as labels in analytical procedures, and incorporated in precursors which are fluorescent only after appropriate chemical treatment.